This invention relates to reagent mixtures useful for detecting and measuring urea.
The determination of urea has been important in the food industry, fertilizer industry, and in clinical chemistry. In clinical chemistry, the determination of urea levels in blood is used routinely as a diagnostic aid in the evaluation of kidney diseases as well as other disease states which are not primarily renal. The method of determining urea in clinical chemistry should ideally be able to measure microgram quantities of urea (due to the low level of urea in serum) with speed, ease, accuracy, and precision. However, the methods of urea analysis normally used in the food and fertilizer industries are far too insensitive for the purposes of clinical chemistry.
The three most widely used methods for the determination of urea in clinical chemistry are the urease-Nessler, urease-Berthelot, and monoximine methods. In the urease-Nessler method, the urea in the unknown specimen is hydrolyzed to carbon dioxide and ammonia. The released ammonia is reacted with mercuric iodide (Nessler's reagent) to give a strong color reaction which is then quantitated colorimetrically. The urease-Berthelot method is similar to the urease-Nessler method except that the released ammonia is reacted with phenol, nitroprusside, and alkaline hypochlorite (Berthelot's reagent) to give the colored reaction. In the monoxime method the urea in the unknown sample is reacted directly with monoxime to give a strong colored complex in the presence of heat and acid.
All of these methods suffer several distinct drawbacks. They include deproteinization, removal of ammonia, nonlinear calibration curves over the clinically useful range, time consuming reaction times and elevated reaction conditions. The enzymatic methods have the advantage of being highly specific due to the specificity of the enzyme urease for urea. They however have the drawbacks of being time consuming, cumbersome to perform, employing toxic reagents, and being highly sensitive to ammonia contamination. The monoxime method has the disadvantage of utilizing toxic chemicals and requiring elevated reaction temperatures up to 100.degree.C to develop the color complex in addition to being less specific than the urease methods.
Another method for the determination of urea involves the use of urease, a buffer, and a pH indicator dye. In such a reaction system, since two moles of NH.sub.3 are formed per mole of CO.sub.2 upon hydrolysis of urea, the net reaction mixture becomes alkaline. The net increase in alkalinity is determined visually with the aid of a pH indicator dye either 1) titrimetrically or 2) by visual comparison with standard color charts. The former method suffers the disadvantage of being lengthy and cumbersome. The latter method suffers the disadvantage of being at best semiquantitative.
I have discovered a new way of determining urea in biological fluids which overcomes the drawbacks of the other methods just mentioned. That is, it is simpler and faster to perform than the urease methods mentioned above, does not require elevated temperatures for reaction, utilizes harmless chemicals, and is quantitative. The method is conveniently carried out at room temperature and the entire reaction is completed in five minutes.
In the reaction mixture urease catalyzes the reaction: EQU Urea + 2H.sub.2 O.fwdarw.2NH.sub.3 + CO.sub.2
only liberated NH.sub.3 is detected in a new unique manner.